Next generation monoclonal antibodies and their mechanisms of action against B-cell lymphomas

68 Pages·2017·1.5 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2012
Next generation monoclonal antibodies and their
mechanisms of action against B-cell lymphomas
Delila Peri
University of Iowa
Copyright 2012 Delila Peri
This thesis is available at Iowa Research Online: https://ir.uiowa.edu/etd/3367
Recommended Citation
Peri, Delila. "Next generation monoclonal antibodies and their mechanisms of action against B-cell lymphomas." MS (Master of
Science) thesis, University of Iowa, 2012.
https://doi.org/10.17077/etd.l53vpmjl
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theImmunology of Infectious Disease Commons
NEXT GENERATION ANTI-CD20 MONOCLONAL
ANTIBODIES AND THEIR MECHANISMS OF ACTION AGAINST
B-CELL LYMPHOMAS
by
Delila Peri
A thesis submitted in partial fulfillment
of the requirements for the Interdisciplinary Studies-Master of
Science degree in Immunology
in the Graduate College of
The University of Iowa
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July 2012

Thesis Supervisor: Professor George Weiner
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER’S THESIS
_______________
This is to certify that the Master’s thesis of
Delila Peri
has been approved by the Examining Committee for the thesis
requirement for the Interdisciplinary Studies-Master of Science degree
in Immunology at the July 2012 graduation.
Thesis Committee: ______________________________
George Weiner, Thesis Supervisor
______________________________
David Lubaroff
______________________________
Siegfried Janz
ACKNOWLEDGMENTS
First I would like to thank my darling children Jonathan and Elizabeth, who
have brought light into my life. I would also like to thank my beloved husband
Moshe for his support throughout my time in the program, as well as my parents
Jerry and DeAnna Kern for their endless support of me.
Additionally I would like to thank my advisor Dr. George Weiner for his
support and patience, as well as my thesis committee and the University of Iowa
Immunology Program.
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ABSTRACT
Next generation monoclonal antibodies (mAbs) are unique in that they are
specifically designed to enhance their mechanisms of action, primarily
complement fixation and antibody-dependent cellular cytotoxicity (ADCC).
Recent studies suggest that complement-fixing properties of a mAb can counter
its ability to activate NK cells and mediate ADCC. GA101, a third generation
(type II anti-CD20) mAb, and rituximab-MAGE (glyco-engineered type I mAb)
show enhanced ADCC and direct cell killing; while ofatumumab, a second
generation anti-CD20 mAb, shows enhanced complement-mediated cytotoxicity
(CMC). These studies set out to determine the primary mechanisms of actions of
these various mAbs, and compare the effect of complement on their ability to
activate NK cells and mediate ADCC or CMC. We also studied the efficiency of
rituximab vs. rituximab-MAGE to deplete B-cells in vivo in mice expressing
human transgenic CD20. In vitro, rituximab and ofatumumab fixed more
complement and mediated a greater degree of CMC, than GA101 and rituximab-
MAGE. Additionally, complement inhibited the ability of both rituximab and
ofatumumab to bind to and activate NK cells, whereas, addition of complement to
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GA101 or rituximab-MAGE did not affect their NK cell activating ability.
Complement also blocked rituximab-induced NK-cell mediated ADCC, but not
GA101-induced NK-cell mediated ADCC. Finally, GA101 and rituximab-MAGE
depleted a higher percentage of B cells in whole blood compared to rituximab
and ofatumumab, whereas rituximab-MAGE depleted fewer B cells, in vivo, in a
complement-dependent fashion. We conclude from these studies that there are
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significant differences among these antibodies and that the ability of a given
antibody to mediate CMC and complement fixation correlates with the ability of
complement to block the interaction between the antibody and NK cells.
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TABLE OF CONTENTS
LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES .............................................................................................. viii
LIST OF ABBREVIATIONS .................................................................................. ix
CHAPTER
I. INTRODUCTION .................................................................................. 1

B cell lymphomas and current therapies ....................................... 1
Antibody structure and anti-CD20 monoclonal antibodies ............ 4
Third generation monoclonal antibodies ....................................... 7
Mechanisms of action for antibody therapy ................................... 9

II. MATERIALS AND METHODS ........................................................... 13

Cell lines, antibodies and serum ................................................. 13
C3b deposition assay .................................................................. 13
Complement mediated cytotoxicity 51Cr release assay ............... 14
Complement mediated propidium iodide assay .......................... 15
NK cell adhesion assay ............................................................... 15
NK cell activation ......................................................................... 16
Antibody dependent cellular cytotoxicity 51Cr release assay ....... 16
B cell depletion assay .................................................................. 17
Mouse studies ............................................................................. 18
Statistical analysis ....................................................................... 18
III. THE COMPARISON OF RITUXIMAB TO VARIOUS SECOND
AND THIRD GENERATION MONOCLONAL ANTIBODIES AND
THEIR MECHANISMS OF ACTION ................................................... 19

Introduction .................................................................................. 19
Differences in complement concentrations are highly 5

variable between various compartments ..................................... 19
Rituximab and ofatumumab have superior complement
fixation properties as compared to GA101 and rituximab-
MAGE .......................................................................................... 21
Rituximab and mediates CMC of Raji cells while GA101
does not mediate CMC ................................................................ 22
Serum inhibitis binding of NK cells to rituximab but not to
GA101 ......................................................................................... 22
GA101 is superior to rituximab and ofatumumab in activating
NK cells in the presence of complement ..................................... 23
C5-depleted serum blocks ADCC mediated by rituximab, but
not mediated by GA101 ............................................................... 24
GA101 and rituximab-MAGE have superior B cell depleting
properties as compared to ofatumumab and rituximab ............... 25
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Rituximab depleted more CD20+ B cells than rituximab-
MAGE in an in vivo model ........................................................... 26
IV. DISCUSSION AND FUTURE DIRECTIONS ...................................... 28

REFERENCES .................................................................................................... 53
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LIST OF TABLES
Table 1. Description of anti-CD20 monoclonal antibody therapies used in these
studies…………………………………………………………………………………..36

Table 2. Complement fixation properties of type I and type II mAbs used in these
studies…………………………………………………………………………………..37
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LIST OF FIGURES
Figure
1. Immunoglobulin structure depicting sugar residues .................................... 34
2. Schematic representation of the interaction between complement,
mAbs, the FcγRIIIa, and the primary mechanism of action used ................ 35
3. Complement levels vary between various compartments ........................... 38
4. Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101 ................................................................. 39
5. Rituximab and ofatumumab have increased complement fixation
capabilities compared to GA101 at various concentrations ........................ 40
6. GA101 and rituximab-MAGE have decreased abilities to fix
complement ................................................................................................. 41
7. Rituximab-MAGE and GA101 have decreased complement fixation
properties in comparison to rituximab alone ................................................ 42
8. GA101 induces lower complement mediated cytotoxicity in the
presence of serum using a 51Cr release assay ........................................... 43
9. GA101 induces lower complement mediated cytotoxicity in the
presence of serum ....................................................................................... 44
10. NK cells adhere to GA101 but not rituximab in the presence of serum ....... 45
11. Complement blocks ability of rituximab coated target cells to activate
NK cells, but not GA101coated Raji cells to activate NK cells .................... 46
12 Complement blocks the ability of rituximab and ofatumumab coated
target cells to activate NK cells, but not GA101 coated cells to activate
NK cells ....................................................................................................... 47
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13. GA101 has enhanced ADCC in the presence of C5-depleted serum ......... 48
14. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab .......................................................................... 49
15. GA101 has superior B cell depleting capabilities compared to
rituximab and ofatumumab at various concentrations ................................. 50
16. Rituximab-MAGE shows superior ability to deplete B cells compared to
rituximab alone ............................................................................................ 51
17. Rituximab depletes more circulating B cells in vivo than rituximab-
MAGE .......................................................................................................... 52
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